Method for treatment of renal failure and occlusive lesion of blood vessels by administration of hepatocyte growth factor

ABSTRACT

The invention presents a preparation for continuous intravenous administration containing hepatocyte growth factor (HGF) as an active ingredient. The preparation for continuous administration of the invention is effective at a lower dose as compared with single or frequent bolus administration of HGF, and therefore side effects can be reduced.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP98/05470 which has an Internationalfiling date of Dec. 3, 1998, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a pharmaceutical preparation forcontinuous intravenous administration containing hepatocyte growthfactor as an active ingredient.

PRIOR ART

Hepatocyte growth factor (HGF) is discovered as a potent proliferationpromoting factor for mature hepatocytes, and is a protein whose gene hasbeen cloned by gene cloning (Biochm Biophys Res Commun, 122, 1450, 1984;Proc. Natl. Acad. Sci, USA, 83, 6489, 1986; FEBS Letter, 224, 311, 1987;Nature 342, 440, 1989; Proc. Natl. Acad. Sci, USA, 87, 3200, 1990).Later studies disclosed that HGF, in vivo, not only works to repair andregenerate damaged liver as liver regenerating factor, but also hasvarious pharmacological actions, and it is expected to be developed asmedicine for kidney diseases, cerebral and neural injuries, cartilageinjuries, arterial diseases, fibroid lung and others, aside from liverdiseases. For example, as for the pharmacological action of HGF on renaldiseases, more specifically, HGF is known to play a key role inregeneration of kidneys. In rats with one kidney removed, an expressionof HGF mRNA was induced in the remaining kidney, and HGF activity waselevated (J. Biol. chem., 266, 22781, 1991). In kidneys of renalischemic rats, HGF mRNA was induced and HGF activity was elevated(American Journal of Physiology, 265, 61, 1993), and in kidneys of miceinjured by a nephrotoxin, similarly, induction of expression of HGF mRNAand elevation of HGF activity were reported (Nephron 73, 735, 1996).Therefore HGF is believed to express and function as a repair factor inrenal injuries.

On the basis of such function of HGF, HGF was administered in renalinjury models, and effects of HGF were reported. That is, since theabove-mentioned renal ischemic models and renal injury models bynephrotoxins such as mercury (II) chloride (HgCl₂) and cisplatin presentacute tubulorrhexis which is a pathology of acute renal failure, theyhave been traditionally used as disease models of acute renal failure.As a result of administration of HGF in such renal ischemic models andrenal injury models, elevation of parameters of renal function disorderssuch as blood urea nitrogen (BUN) and blood creatinine was promptlysuppressed in both injury models, and it was unveiled that HGF has arepair action on renal injuries (American Journal of Physiology, 266,129, 1994; Proc. Natl. Acad. Sci, USA, 91, 4357, 1994).

In addition to these acute renal failure models such as renal ischemicmodels and renal injury models, in spontaneous nephrotic models regardedas chronic renal failure models, HGF is also found to present markedeffects (Japan Disease Model Academy Record Vol. 13, 113, Lecture 28,1997).

Thus, as a result of evaluation of effects of HGF in various animalmodels, the efficacy of HGF in renal diseases ranging from acute renalfailure to chronic renal failure has been elucidated. However, the bestadministration route and dose of HGF has not been known.

Generally, it is common knowledge that a protein preparation isintravenously administered. Concerning intravenous administration of HGFto renal disease models, for example, frequent intravenousadministrations (CYTOKINE, 8, 387, 1996; Proc. Natl. Acad. Sci, USA, 91,4357, 1994) and single intravenous administrations (J. American Societyof Nephrology, 1835, Lecture A2944, 1996; J. of Japan Society ofNephrology, Vol. 39, 260, Lecture 0-302) are reported. However,continuous intravenous administration of HGF in renal disease models hasnot been reported yet.

The present inventors intensively studied the dose and route ofadministration for applying HGF to renal disease. As method ofadministration of HGF for renal disease, intravenous bolusadministration and continuous intravenous administration wereinvestigated, and the intravenous bolus administration is established inevaluation, whereas the continuous intravenous administration is notestablished in evaluation, because 1) the cannula is often pulled outduring continuous administration, 2) it takes much time in operation inthe case of cannulation into the cervical vein although the cannula isless likely to be pulled out, and 3) pathological control is difficultto due stress to animals, among other problems. In such background, sofar, attempts of continuous intravenous administration in renal diseasemodels have not been reported, and hence it is not known whether thecontinuous intravenous administration of HGF is effective or not.

In such circumstances, the inventors have succeeded in establishment ofevaluation system of continuous intravenous administration in mercurychloride model mice by making some improvements, including indwell ingof wing-shaped needle in tail vein instead of cervical vein, andanesthesia of mice. Using this model, effects of continuous intravenousadministration of HGF were studied actually, and notable effects of HGFby continuous administration, that is, effects of continuous intravenousadministration of HGF on renal disease have been disclosed for the firsttime.

The inventors further compared the effects between intravenous bolusadministration and continuous intravenous administration, anddiscovered, surprisingly enough, that the dose can be substantiallyreduced in continuous administration as compared with bolusadministration. Since the dose is decreased as compared with theconventional bolus administration, it means that side effects aredecreased in clinical use.

On the basis of these findings, the inventors have further promotedresearches, and found that the continuous intravenous administration ofHGF is more effective than bolus administration on occlusive lesion ofblood vessels. More specifically, in the glycerol model foradministering glycerol in muscles, it is accompanied by injury ofskeletal muscles, a large quantity of myoglobin is released fromskeletal tissues into blood, and various components such as creatinekinase, creatine, potassium, phosphoric acid and purine precursor arereleased into the blood. Thus, the glycerol model is regarded as theso-called MNMS (myonephropathic-metabolic syndrome) model such asrhabdomyolysis, myoglobinuria and detrition syndrome that areaccompanied by decay of skeletal tissues (Syndrome by Clinical Regionsof Japan, Supplement 17, Nephrotic Syndrome, pp. 523-526, 1997, etc.).

On the other hand, in occlusive lesion of blood vessels, if ischemiacontinues due to acute vascular occlusion, ischemic muscular necrosiswidely occurs, and MNMS that elevates levels of creatine kinase,potassium and myoglobin in serum is induced. Therefore, the glycerolmodel is regarded also as a model of occlusive lesion of blood vessel.In such glycerol model, too, the continuous intravenous administrationof HGF is more effective than the bolus administration, and thecontinuous intravenous administration of HGF has been proved to beeffective also on occlusive lesion of blood vessel.

As a result of further studies, the inventors have found that thecontinuous intravenous administration of HGF is more effective than thebolus administration in platelet increasing action of HGF, and generallythat the continuous intravenous administration is more effective thansingle or frequent intravenous bolus administration of HGF, and henceconcluded that the effect can be expressed at low dose.

The invention is devised on the basis of these findings, and it is hencean object of the invention- to present a pharmaceutical preparation forcontinuous intravenous administration containing HGF as an activeingredient.

DISCLOSURE OF THE INVENTION

The invention presents a pharmaceutical preparation for continuousintravenous administration containing HGF as an active ingredient. Moreparticularly, it presents a pharmaceutical preparation for continuousintravenous administration effective for treatment and prevent ion ofrenal diseases, occlusive lesion of blood vessels, and others. Thepharmaceutical preparation for continuous intravenous administration ofthe invention is lower in dose as compared with the intravenous bolusadministration, and is hence effective to decrease side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing an outline of administration schedule ofExample 1-3).

FIG. 2 is a graph showing an influence of anesthesia on the presentevaluation system, in which A represents the influence on BUN, and Brepresents the influence on creatinine.

FIG. 3 is a diagram showing an outline of administration schedule ofExample 1-4).

FIG. 4 is a graph showing effects of continuous intravenousadministration of HGF 200 μg/kg/hr (1000 μg/kg/day) from 0.5 hr after to5.5 hr after administration of HgCl₂, in which A represents the effecton BUN, and B represents the effect on creatinine.

FIG. 5 is a diagram showing an outline of administration schedule ofExample 2-2).

FIG. 6 is a graph showing effects of continuous intravenousadministration of HGF 60 μg/kg/hr (300 μg/kg/day) and HGF 200 μg/kg/hr(1000 μg/kg/day) from 0.5 hr after to 5.5 hr after administration ofHgCl₂, in which A represents the effect on creatinine, and B representsthe effect on BUN.

FIG. 7 is a diagram showing an outline of administration schedule ofExample 2-3).

FIG. 8 is a graph showing effects of continuous intravenousadministration of HGF 60 μg/kg/hr (120 μg/kg/day) and HGF 200 μg/kg/hr(400 μg/kg/day) from 0.5 hr after to 2.5 hr after administration ofHgCl₂, in which A represents the effect on creatinine, and B representsthe effect on BUN.

FIG. 9 is a diagram showing an outline of administration schedule ofExample 2-4).

FIG. 10 is a graph showing effects of intravenous bolus administrationof HGF (100 μg/kg (group 2), 300 μg/kg (group 3), 1000 μg/kg (group 4),and 3000 μg/kg (group 5)) at 0.5 hr after administration of HgCl₂, inwhich A represents the effect on creatinine, and B represents the effecton BUN.

FIG. 11 is a diagram showing an outline of administration schedule ofExample 3-2).

FIG. 12 is a graph showing effects of continuous intravenousadministration of HGF 60 μg/kg/hr (180 μg/kg/day) and HGF 200 μg/kg/hr(600 μg/kg/day) from 0.5 hr after to 3.5 hr after administration ofHgCl₂, in which A represents the effect on creatinine, and B representsthe effect on BUN.

FIG. 13 is a graph showing increase of platelet counts by intravenousadministration of HGF, in which A represents continuous intravenousadministration of HGF (mean±SD, n=3, **: p<0.01 to control), and Brepresents intravenous bolus administration of HGF (mean±SD, n=6, **:p<0.01 to control).

BEST MODE FOR CARRYING OUT THE INVENTION

HGF used in the invention is a known substance, compounds prepared byvarious methods can be used if they are purified to an extent that theymay be used as a medicine, or any commercial product can be used (forexample, Toyobo Code No. HGF-101). To prepare HGF, for example, primaryculture cells or a cell line which produce(s) HGF are cultivated, andHGF is obtained by isolating from the culture supernatant and purifying.Or by gene engineering technique, a gene encoding HGF is inserted into aproper vector, and it is incorporated into a proper host to transform,and a desired recombinant HGF is obtained from the culture supernatantof this transformant (for example, Nature, 342, 440, 1989; JapaneseLaid-open Patent No. 5-111383; Biochem. Biophys. Res. Commun. 163, 967,1989). The host cell is not particularly limited, and various host cellsconventionally used in gene engineering technique may be used, whichare, for example. Escherichia coli, yeast, and animal cells. Thusobtained HGF is not limited as far as it has substantially the sameaction as the natural HGF, and for example, one or plural amino acids inthe amino acid sequence may be replaced, deleted and/or added, orsimilarly sugar chain may be replaced, deleted and/or added.

The preparation for continuous intravenous administration of theinvention may be applied in various diseases in which the medicinaleffects of HGF have been recognized, and in particular it is preferablyused in renal diseases and occlusive lesion of blood vessels. The renaldiseases mentioned above include both chronic renal diseases(nephropathy, renal failure, nephritis) and acute renal diseases.Specific examples include acute renal failure, chronic renal failure,glomerulonephritis, nephrotic syndrome, systemic lupus erythematosus,glomerular diseases accompanying hepatic disease, diabetic nephropathy,tubulointerstitial nephritis, renal vascular disorder, hypertensiverenal disorder, nephro-urinary calculus, urinary tract infection,occlusive nephropathy, cystic renal disease, nephro-urinary tumor,hereditary renal disease, kidney transplant, complication by kidneytransplant, and drug-induced renal disorder. Examples of occlusivelesion are lower limb ischemia, acute arterial occlusion, chronicarterial occlusion, arteriosclerosis obliterans, arterial embolism,arterial thrombosis, Buerger's disease, venous occlusion, venousthrombosis, thrombotic phlebitis, angiodysplasia, vascular damage,coronary occlusion, coronary stenosis, pulmonary embolism, and arterialocclusion of organs (Clinical Angiology, Bunkodo, 1992; Therapeutics,Vol. 31, No. 3, pp. 284-338, 1997).

At this time, the active ingredient of HGF may be combined withadditives as required, such as pH regulating agent, buffer, stabilizer,preservative, or solubilizer. The dose varies with the symptom, age, sexand others, and, for example, in the case of treatment or prevention ofrenal disease, a dose of 500 μg/head/hr or less, preferably 60μg/head/hr or less is intravenously injected in 30 minutes to 5 hours,preferably 30 minutes to 3 hours. For treatment or prevention ofocclusive lesion of blood vessel, a dose of 1800 μg/head/hr or less,preferably 200 μg/head/hr or less is intravenously injected in 30minutes to 3 hours, preferably 30 minutes to 1.5 hours.

If the effect is not enough by single continuous administration, thecontinuous administration may be repeated plural times.

The timing of administration of the preparation for continuousintravenous administration of the invention is not particularlyspecified, but as described in the following Examples, in acute renalfailure, notable effects are exhibited by continuous administration ininitial phase of onset, and it is preferred to administer in initialphase of disease of the patient.

The preparation for continuous intravenous administration of theinvention is used for the purpose of prevention, aside from thetherapeutic purpose as mentioned above. For example, acute renal failuredue to ischemic or nephrotoxic cause is the disease taking place as sideeffect of surgical bleeding or administration of antibiotics, anti-tumoragent or contrast medium, and therefore onset of such acute renalfailure may be predicted depending on the degree of bleeding, dose ofantibiotic or contrast medium, or condition of patient. In such a case,the preparation for continuous intravenous administration of theinvention may be used for preventive purpose of acute renal failure.

INDUSTRIAL APPLICABILITY

The invention presents the preparation for continuous intravenousadministration containing HGF as an active ingredient. The preparationfor continuous administration of the invention can reduce dose ascompared with bolus administration, and therefore side effects aredecreased.

EXAMPLES

Preferred Examples of the invention are described below, but it must benoted that the invention is not limited to the illustrated Examplesalone.

Example 1

Study on Continuous Intravenous Administration of HGF in Mouse RenalDisease Model (1)

1) Animals

Male BALB/c mice (5 or 6 weeks old) were purchased from SLC, andpreliminarily raised in the conditions of temperature of 23±2° C.,humidity of 55±10%, lighting from 8:00 to 20:00, and free access to dietand water, and used for experiment at the age of 6.5 weeks to 8 weeks.

2) Intravenous Continuous Administration to Mice

A 1 ml syringe (Terumo Corp.) was preliminarily filled with HGF orvehicle, and a winged needle (Terumo) was attached to it with athree-way stopcock (Terumo) placed between them (then the wing being cutoff). Mice were anesthetized by Nembutal (100 mg/kg, s.c.). The needlewas inserted into the tail vein and kept on a hot plate at 37° C. andfixed with tape. A breathing hole was opened in the bottom of a conicaltube having a proper size for BALB/c mouse, and also a slit forprojecting the tail was opened at the opposite side of the tube, andthen each mouse was put in the tube. The syringes were set in aninfusion pump (Neuro Science Corp.), and piston positions were alignedby the three-way stopcock, and the evaluation system for continuousintravenous administration was set up, and used in the followingexperiments (Examples 1 to 3).

3) Study on Effects of Anesthesia

Since Nembutal is used for anesthesia in this evaluation system asdescribed in 2), influence of anesthesia on model preparation and effectof HGF were investigated in the first place. An outline ofadministration schedule is shown in FIG. 1.

Male BALB/c mice at the age of 8 weeks were divided into four groups of8 animals each, and 8.5 mg/kg of HgCl₂ was subcutaneously administeredin the back in all mice. This time was hour 0. Neither diet nor waterwas given from 12 hours before to 12 hours after HgCl₂ injection. Ingroup 1 and group 2, before administration of HgCl₂, 100 mg/kg ofNembutal was subcutaneously administered in the rear back. At 0.5, 6,12, 24, and 36 hours after administration of HgCl₂, vehicle wasadministered in group 1 and group 3, and HGF in group 2 and group 4. Thedose of HGF was 500 μg/kg/shot. Blood was sampled from all mice 48 hoursafter HgCl₂ injection, and the serum was separated, and the blood ureanitrogen (BUN) and creatinine were measured (using Synchron CX3 SystemDelta by Beckmann). Human recombinant HGF (same in the followingexperiments) was used, and the vehicle was 10 mM citric acid buffer (pH6.0) containing 0.01% Tween 80 and 0.3 M NaCl. Statistical analysis wasby Tukey's test.

Results are shown in FIG. 2. A degree of creatinine elevation was notdifferent notably regardless of anesthesia. By HGF, a significantsuppression of creatinine was observed, but a degree of effect washardly influenced by anesthesia. It was hence known that influence ofanesthesia could be practically ignored when evaluating the effect ofHGF.

4) Evaluation of Effect by Continuous Intravenous Administration

An outline of administration schedule is shown in FIG. 3.

Male BALB/c mice at the age of 8 weeks were divided into two groups of10 animals each, and 8.5 mg/kg of HgCl₂ was subcutaneously administeredin the back in all mice. This time was hour 0. Neither diet nor waterwas given from 12 hours before to 12 hours after HgCl₂ injection.Continuous intravenous administration of vehicle (group 1) or HGF (group2) was performed by using the evaluation system mentioned in 2). Justbefore administration of HgCl₂, mice were anesthetized with Nembutal(100 mg/kg, s.c.), and from 0.5 hr after HgCl₂ injection, theadministration was started. HGF (1000 μg/kg/day) was administeredcontinuously for 5 hours at 200 μg/kg/hr. Blood was sampled from allmice 48 hours after HgCl₂ injection, and the serum was separated, andthe blood urea nitrogen (BUN) and creatinine were measured (usingSynchron CX3 System Delta by Beckmann). Statistical analysis was byt-test.

Results are shown in FIG. 4. Both BUN and creatinine were significantlysuppressed by continuous intravenous administration of HGF. Therefore,it was clarified that continuous intravenous administration of HGF iseffective for renal disease.

Example 2

Study on Continuous Intravenous Administration of HGF in Mouse RenalDisease Model (2)

1) Animals

Male BALB/c mice (6 weeks old) were purchased from SLC, andpreliminarily raised in the conditions of temperature of 23±2° C.,humidity of 55±10%, lighting from 8:00 to 20:00, and free access to dietand water, and used for experiment at the age of 6.5 weeks.

2) Attempt to Reduce the Dose of HGF (1)

Since the efficacy was confirmed in Example 1-4) by administering HGFfor 5 hours at a rate of 200 μg/kg/hr, the rate of administration of HGFwas reduced from 200 μg/kg/hr to 60 μg/kg/hr. An outline ofadministration schedule is shown in FIG. 5.

Male BALB/c mice (6.5 weeks) were divided into three groups of 10animals each, and anesthetized by Nembutal (100 mg/kg, s.c.).Immediately after Nembutal injection 8.5 mg/kg of HgCl₂ wassubcutaneously administered in the back in all mice. This time was hour0. Neither diet nor water was given from 12 hours before to 12 hoursafter HgCl₂ injection. Vehicle was administered in group 1. HGF in group2 at 60 μg/kg/hr (300 μg/kg/day), and HGF in group 3 at 200 μg/kg/hr(1000 μg/kg/day), by continuous intravenous injection from 0.5 to 5.5hours after HgCl₂ injection. Blood was sampled from all mice 48 hoursafter HgCl₂ injection, and the serum was separated, and the blood ureanitrogen (BUN) and creatinine were measured (using Synchron CX3 SystemDelta by Beckmann). The data was statistically processed bynonparametric test because equal variance was denied in the Bartlett'stest.

Results are shown in FIG. 6. Although the efficacy was slightly inferiorto that of the positive control of 200 μg/kg/hr (1000 μg/kg/day), thecreatinine elevation was significantly suppressed at 60 μg/kg/hr (300μg/kg/day). A suppressing tendency was noted in BUN if not significant.

3) Attempt to Reduce the Dose of HGF (2)

The time of continuous administration mentioned in 2) was reduced from 5hours to 2 hours. An outline of administration schedule is shown in FIG.7.

Male BALB/c mice (6.5 weeks) were divided into three groups of 10animals each, and anesthetized by Nembutal (100 mg/kg, s.c.).Immediately after Nembutal injection 8.5 mg/kg of HgCl₂ wassubcutaneously administered in the back in all mice. This-time was hour0. Neither diet nor water was given from 12 hours before to 12 hoursafter HgCl₂ injection. Vehicle was administered in group 1, HGF in group2 at 60 μg/kg/hr (120 μg/kg/day), and HGF in group 3 at 200 μg/kg/hr(400 μg/kg/day), by continuous intravenous injection from 0.5 to 2.5hours after HgCl₂ injection. Blood was sampled from all mice 48 hoursafter HgCl₂ injection, and the serum was separated, and the blood ureanitrogen (BUN) and creatinine were measured (using Synchron CX3 SystemDelta by Beckmann). The data was statistically processed bynonparametric test because equal variance was denied in the Bartlett'stest.

Results are shown in FIG. 8. In this experiment, the BUN and creatininewere not improved significantly, but an improving tendency was noted.

4) Dose-Dependent Effect by Intravenous Bolus Administration of HGF

By way of comparison with continuous intravenous administration, thedose-dependent effect by intravenous bolus administration of HGF wasinvestigated. An outline of administration schedule is shown in FIG. 9.

Male BALB/c mice were divided into five groups of 10 animals each, and8.5 mg/kg of HgCl₂ was subcutaneously administered in the back in allmice. This time was hour 0. Neither diet nor water was given from 12hours before to 12 hours after HgCl₂ injection. In group 1, vehicle wasadministered in the tail vein 0.5 hour after HgCl₂ injection. In groups2 to 5, HGF (100, 300, 1000, 3000 μg/kg, respectively) was administeredin the tail vein 0.5 hour after HgCl₂ injection. Blood was sampled fromall mice 48 hours after HgCl₂ injection, and the serum was separated,and the blood urea nitrogen (BUN) and creatinine were measured (usingSynchron CX3 System Delta by Beckmann). The data was statisticallyprocessed by Dunnett's test.

Results are shown in FIG. 10. The creatinine: level was significantlyimproved at dose of 1000 μg/kg/day or more, but only an improvingtendency was noted at 300 μg/kg/day, and no effect was observed at 100μg/kg/day. By contrast, by continuous intravenous administration, asignificant improvement was recognized at 300 μg/kg/day (see 2 above),and an improving tendency was noted at 120 μg/kg/day (see 3 above). As aresult, as compared with the single intravenous administration, it wasfound that the dose could be saved in the continuous intravenousadministration. The inventors have also compared the effect betweenfrequent intravenous administration and single shot by adjusting thetotal dose, and reported that similar results are obtained in singleshot and frequent administration (J. of Japan Society of Nephrology.Vol. 39, 260. Lecture 0-302). Therefore, the continuous intravenousadministration of HGF can decrease the dose as compared with single shotor frequent intravenous bolus administration.

Example 3

Study on Continuous Intravenous Administration of HGF in Mouse RenalDisease Model (3)

1) Animals

Male BALB/c mice were purchased at the age of 6 weeks from SLC, andpreliminarily raised in the conditions of temperature of 23±2° C.,humidity of 55±10%, lighting from 8:00 to 20:00, and free access to dietand water, and used for experiment.

2) Attempt to Reduce the Dose of HGF

In Example 2-2), although the dose was reduced to 300 μg/kg/day, but at120 μg/kg/day, only an improving tendency was noted, and significantimprovement was not obtained. It was hence attempted to study at anapproximately intermediate dose of 180 μg/kg/day. An outline ofadministration schedule is shown in FIG. 11.

Male BALB/c mice (6.5 weeks) were divided into three groups of 10animals each, and anesthetized by Nembutal (100 mg/kg, s.c.).Immediately after Nembutal injection 9 mg/kg of HgCl₂ was subcutaneouslyadministered in the back in all mice. This time was hour 0. Neither dietnor water was given from 12 hours before to 12 hours after HgCl₂injection. Vehicle was administered in group 1, HGF in group 2 at 60μg/kg/hr (180 μg/kg/day), and HGF in group 3 (positive control) at 200μg/kg/hr (600 μg/kg/day), by continuous intravenous injection from 0.5to 3.5 hours after HgCl₂ injection. Blood was sampled from all mice 48hours after HgCl₂ injection, and the serum was separated, and creatinineand blood urea nitrogen (BUN) were measured (using Synchron CX3 SystemDelta by Beckmann). The data was statistically processed bynonparametric test because equal variance was denied in the Bartlett'stest.

Results are shown in FIG. 12. In HGF 180 μg/kg/day group, elevation ofcreatinine and BUN was significantly suppressed as compared with vehiclegroup. Its suppression was nearly same as positive control (HGF 600μg/kg/day group).

Example 4

Study on Continuous Intravenous Administration of HGF in Rat GlycerolModel

1) Materials and Method

Male Wistar rats at the age of 7 to 8 weeks (weighing about 200 g) wereused. Water was withheld form 15 hours before to 8 hours after glycerolinjection. 50% glycerol was intramuscularly administered (10 ml/kg)under ether anesthesia. An intravenous continuous infusion of HGF (1mg/kg, 30 minutes) or bolus administration of HGF (3 times, total 750μg/kg) was started 8 hours after glycerol injection. The survival of therats was observed for 11 days after the administration of glycerol.

2) Results

Survival rate on 11th day after glycerol administration was 2/8 incontrol group and 8/10 in HGF group by continuous administration. Bychi-square test, HGF significantly elevated the survival rate (P<0.05).In the case of bolus administration, the survival rate was 5/8 incontrol group and 5/8 in HGF group, and the life-saving effect by HGFwas not recognized. It was therefore found that continuousadministration tends to be more beneficial than bolus administration.

Thus, in rat glycerol models, it is revealed that continuous intravenousadministration of HGF is more effective than bolus administration.Therefore, in occlusive lesion of blood vessels expressing MNMS that issimilar to glycerol models, it is found that continuous intravenousadministration of HGF is more effective than intravenous bolusadministration.

Example 5

Study on Continuous Intravenous Administration of HGF for Increase ofPlatelet Counts

1) Materials and Method

Male Fisher rats of 7 to 8 weeks of age were used. In continuousadministration, HGF was diluted to 2 ml/kg/2 hr with vehicle of PPScontaining 0.01% of Tween 80 and 0.25% of HAS, and administered in tailvein. A single dose was 0.25 mg/kg, and only vehicle was administered incontrol group. The administrations were carried out for 7 consecutivedays at the rate of twice a day, blood was sampled next day after thefinal administration, and platelets were counted by using asemiautomatic blood cell count analyzer (Sysmex F-800).

In bolus administration, HGF was diluted to 1 ml/kg/shot with vehicle,and administered in caudal vein. Single doses were 0, 0.125, 0.25, 0.5,1, and 2 mg/kg, which were administered for 7 consecutive days at therate of twice a day.

2) Results

Results are shown in FIG. 13. In the case of bolus administration,platelet counts were significantly increased at dose of 0.5 mg/kg×2/dayor more. In continuous administration, platelet counts weresignificantly increased at dose of 0.25 mg/kg×2/day. Hence, bycontinuous administration, platelet counts were increased at a lowerdose.

Preparation Example 1

A solution containing 1 mg of HGF, 1 g of mannitol, and 10 mg ofpolysorbate 80 in 100 ml of physiological saline was asepticallyprepared, and 1 ml aliquot of the solution were poured into vialsseparately. They were lyophilized and sealed to obtain lyophilizedpreparations.

Preparation Example 2

A solution containing 1 mg of HGF and 100 mg of human serum albumin in100 ml of 0.02M Phosphate buffer (containing 0.15M NaCl and 0.01%polysorbate 80; pH 7.4) was aseptically prepared, and 1 ml aliquot ofthe solution were poured into vials separately. They were lyophilizedand sealed to obtain lyophilized preparations.

1. A method for treating renal failure, which comprises administering aneffective amount of hepatocyte growth factor (HGF) by continuousintravenous administration to suppress blood urea nitrogen (BUN) andcreatinine levels in a patient suffering from renal failure, therebytreating renal failure in said patient.